Acetylcholine receptor based chemogenetics engineered for neuronal inhibition and seizure control assessed in mice

Epilepsy is a prevalent disorder involving neuronal network hyperexcitability, yet existing therapeutic strategies often fail to provide optimal patient outcomes. Chemogenetic approaches, where exogenous receptors are expressed in defined brain areas and specifically activated by selective agonists, are appealing methods to constrain overactive neuronal activity. We developed BARNI (Bradanicline- and Acetylcholine-activated Receptor for Neuronal Inhibition), an engineered channel comprised of the α7 nicotinic acetylcholine receptor ligand-binding domain coupled to an α1 glycine receptor anion pore domain. Here we demonstrate that BARNI activation by the clinical stage α7 nicotinic acetylcholine receptor-selective agonist bradanicline effectively suppressed targeted neuronal activity, and controlled both acute and chronic seizures in male mice. Our results provide evidence for the use of an inhibitory acetylcholine-based engineered channel activatable by both exogenous and endogenous agonists as a potential therapeutic approach to treating epilepsy.

Supplementary Fig. 1: Amino acid sequence of the BARNI channel.

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Validation of BARNI channel efficacy in dissociated hippocampal neurons.
channel activation dose response in acute hippocampal slices.

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Impacts of BARNI channel on memory-associated hippocampal oscillations.Hippocampal local field potential (LFP) was recorded with implanted 4-channel silicon probes as mice freely explored an open field.Representative LFP frequency spectra examples from Scramble-and BARNI-expressing animals during the baseline 30 min following intraperitoneal saline injection (a) and 30 min post-bradanicline (100 mg/kg) injection phases (b).Head-mounted accelerometry tracked bouts of elevated active movement (>5 sec).Periods of theta-filtered (90-200 Hz) signal at baseline (c) and post-bradanicline (d) from active periods within the above examples.e) Activity-associated rhythm band power was not altered at baseline between construct groups in the theta (P=0.999),low gamma (P=0.598) or high gamma (P=0.707)frequency bands.f) There were also no construct-associated within animal shifts from baseline in theta (P=0.189),low gamma (P=0.744) or high gamma (P=0.718)amplitudes post-bradanicline.Representative sharp wave-ripples, filtered at 90-200 Hz, occurring during the baseline (g) and post-bradanicline (h) periods of the above examples.i) At baseline, there were no differences between Scramble-and BARNI-expressing animals in the non-active LFP properties of ripple amplitude (P=0.574),duration (P=0.647),intra-ripple frequency (P=0.056) or rate of ripple occurrence (P=0.272).j) Post-bradanicline within animal shifts did not differ between groups in ripple amplitude (P=0.431),duration (P=0.288) or intra-ripple frequency (P=0.825),rate of ripple occurrence (P=0.115).Between group differences were assessed with two-sided t-tests.Data are presented as Mean ± SEM. n = 4 animals per group.Source data are provided as a Source Data file.SupplementaryFig.9: Absolute quantifications of seizure frequency and duration.a) Representative seizure with insets showing spike frequency at the beginning (green) and towards the end (purple) of the seizure.b) Top, frequency of spontaneous seizures decreased after a single intraperitoneal (i.p.) injection of bradanicline (Br; 100 mg/kg).30 minute bins.(F(33, 374)=3.322,P<0.0001, two-way RM ANOVA, Time x Vector + Drug).Bottom, brief change in the duration of spontaneous seizures observed after a single i.p. injection of bradanicline.30 minute bins.(F(33, 374)=2.7,P<0.0001, two-way RM ANOVA, Time x Vector + Drug).Significance values on graph are shown for comparison between vehicle and bradanicline treatment in BARNI-expressing mice at each time point using paired two-sided t-test.Significance values in legend are shown for comparison between vector expression and treatment groups using a two-sided Tukey's multiple comparisons test.Mean ± SEM, n = 9 Scramble, 10 BARNI mice.*P<0.05,**P<0.01,****P<0.0001.Source data are provided as a Source Data file.Seizure analysis using 6-second seizure duration threshold.Top, frequency of spontaneous seizures decreased after a single intraperitoneal (i.p.) injection of bradanicline.30 minute bins.(F(33, 198)=1.951,P=0.0028, two-way RM ANOVA, Time x Vector + Drug).Bottom, duration of spontaneous seizures observed after a single i.p. injection of bradanicline (Br; 100 mg/kg).30 minute bins, dashed line indicates time of second dose.(F(33, 198)=1.809,P=0.0072, two-way RM ANOVA, Time x Vector + Drug).Significance values on graph are shown for comparison between vehicle and bradanicline treatment in BARNI-expressing mice at each time point using paired two-sided t-test.Significance values in legend are shown for comparison between vector expression and treatment groups using a two-sided Tukey's multiple comparisons test.Mean ± SEM, n = 6 Scramble, 5 BARNI mice.*P<0.05,**P<0.01,****P<0.0001.Source data are provided as a Source Data file.Supplementary Figure 11: Repeat dosing on seizure frequency and duration.a) Top, prolonged decrease in frequency of spontaneous seizures after two intraperitoneal (i.p.) injections of the same compound of saline or bradanicline (Br; 100 mg/kg) spaced 2 hours apart.30 minute bins, dashed line indicates time of second dose.(F(63, 378)=4.553,P<0.0001, two-way RM ANOVA, Time x Vector + Drug).Bottom, brief change in the duration of spontaneous seizures observed after the first of two i.p. injections of the same compound spaced 2 hours apart.30 minute bins, dashed line indicates time of second dose.(F(63, 378)=2.579,P<0.0001, two-way RM ANOVA, Time x Vector + Drug).Significance values on graph are shown for comparison between vehicle and bradanicline treatment in BARNI-expressing mice at each time point using paired two-sided t-test.Significance values in legend are shown for comparison between vector expression and treatment groups using a two-sided Tukey's multiple comparisons test.Mean ± SEM, n = 6 Scramble, 5 BARNI mice.*P<0.05,**P<0.01,***P<0.001,****P<0.0001.b) Normalized seizure frequency 5 hours after first bradanicline dose in a 2-dose drug regimen (second dose administered 2 hours after the first) across several days in a BARNIexpressing mouse.Source data are provided as a Source Data file.
Decreasing cumulative number of seizures in BARNI-expressing mice over time.Baseline recordings were taken starting 1-2 days after virus injection.Cumulative number of seizures over an approximately 3.5 week time period from the start of recording is shown, normalized to the cumulative number of seizures observed at 14 days of recording for each mouse.BARNI-expressing mice show a lower cumulative number of seizures at the end of the baseline recording period compared to Scramble-expressing mice (Mixed-effects model, Time x Vector: F (24, 338) = 3.518, P<0.0001).Mean ± SEM, n = 8 Scramble, 10 BARNI mice.****P<0.0001.Source data are provided as a Source Data file.

6: Bradanicline suppression of neuronal excitability requires receptors with α7 nACh ligand-binding domains.
Current clamp recordings of BARNI or Scramble-expressing transduced CA1 neurons in acute hippocampal slices in aCSF containing 0.25 µM α-Bungarotoxin (BTX).BTX is a selective antagonist of both endogenous α7 nAChRs and the α7 nACh ligand binding domaincontaining BARNI channel.a) Left, time course of input resistance values in response to 1 min bath application of bradanicline (BR; 0.15 µM), sampled every 5 sec.Right, Time-binned input resistance values for individual animals (small circles) and within groups (large circles) were unchanged in BARNI or Scramble-expressing neurons by bradanicline application (F(2,27)=0.88,P=0.427).b) Left, time course of measured rheobase currents required to evoke action potentials in response to 1 min bath application of bradanicline.Right, Time-binned rheobase values were equivalent between BARNI vs Scramble-expressing neurons in response to bradanicline application (F(2,26)=0.01,P=0.991).c) Left, time course of action potential (AP) counts, normalized to the pre-drug average for each neuron, in response to 1 min bath application of bradanicline.Right, Time-binned action potential counts remained similar in BARNI vs Scrambleexpressing neurons after bradanicline application (F(2,27)=0.29,P=0.753).Baseline values are averaged across 1 minute prior to drug c Supplementary Fig. application, bradanicline values are 1-3 minutes after application and Wash values are > 8 minutes after drug.Mean ± SEM, n = 7 cells/2 animals BARNI, n = 4 cells/2 animals Scramble.*P<0.0%(two-way ANOVA with two-sided Tukey's HSD).Source data are provided as a Source Data file.

Quantifying bradanicline concentrations in plasma and brain tissue following in vivo dosing.
Measurements of bradanicline concentrations detectable in plasma (in ng/ml) and in total brain tissue (in ng/g) following an initial administration via oral gavage.b) Plasma concentrations of bradanicline measured after an intraperitoneal (i.p.) administration and used to calculate estimates of brain concentrations based on observed plasma-to-brain ratios following oral administration.All measurements were made using LC-MS/MS.Dotted lines in each plot indicate the EC 50 for BARNI channel activation recorded in dissociated hippocampal neurons.Mean ± SEM, n = 3 animals per time point.Source data are provided as a Source Data file. a)